Immunotoxins in which monoclonal antibodies or ligands against overexpressed proteins on the surface of cancer cells are conjugated to plant or bacterial toxins, have been extensively investigated for their possible use as anticancer agents (Non-Patent Document 1). A number of immunotoxins have been tested in preclinical and clinical trials, and interleukin-2-diphteria toxin (IL2-DT; Ontak™) has been approved for the treatment of chronic T cell lymphocytic leukemia (CLL) (Non-Patent Document 2, Non-Patent Document 3). In addition, Pseudomonas exotoxin-based immunotoxins including interleukin-4-Pseudomonas exotoxin [IL4 (38-37)-PE38 KDEL] and interleukin-13-Pseudomonas exotoxin (IL13-PE38QQR) have been tested in clinical trials (Non-Patent Document 4, Non-Patent Document 5). Both Diphtheria toxin and Pseudomonas exotoxin act by catalytically inactivating the elongation factor 2 protein in the ribosome complex, after uptake into lysosomes, activation and translocation into the cytosol. This mechanism of action allows the immunotoxins to efficiently destroy dormant, non-replicating tumor cells.
Although the targeting approach towards cancer utilizing bacterial toxin-based immunotoxin is fascinating, its limitation of use lies in the liver toxicity due to the bacterial toxin and immunogencity caused by the toxin proteins (Non-Patent Document 2, Non-Patent Document 4, Non-Patent Document 6). In addition, molecular size of immunotoxins is generally larger compared to chemical compounds or fragment antibody drugs, which might prevent drugs from efficiently penetrating into tumor mass in the human body. To overcome these issues, new generation immunotoxins with evolutional approach are critically needed.
Epidermal growth factor receptor (EGFR) has been an important tumor-specific target for many years (Non-Patent Document 7, Non-Patent Document 8). EGFR plays important roles in cellular growth, differentiation, and migration. Its positive signaling was found to cause increased proliferation, decreased apoptosis, and enhanced tumor cell motility and angiogenesis (Non-Patent Document 9). EGFR overexpression has been frequently found in a wide spectrum of human tumors of epithelial origin, including breast, lung, gastric, colorectal, prostate, pancreatic and ovarian cancers (Non-Patent Document 10). All these findings have shown that EGFR is important as a target for receptor-mediated delivery system of drugs. Recently, several studies have reported the successful identification of peptide ligands of EGFR by screening phage display libraries, implicating possible drug delivery targeting EGFR (Non-Patent Document 11, Non-Patent Document 12).
Therapeutic peptides are increasingly gaining popularity in use in a variety of applications (for example, tumor vaccine (Non-Patent Document 13), antimicrobial therapy (Non-Patent Document 14), and nucleic acid delivery (Non-Patent Document 15)) (Non-Patent Document 16). In addition, research and development of new cancer therapy involving peptide-based drug has been undertaken (Non-Patent Document 17, Non-Patent Document 18). It is also known that peptide therapeutics are relatively easily generated using either recombinant or solid-phase chemical synthesis techniques and are generally less expensive when compared to antibody-based therapeutics. In recent years, it has been reported that a new lytic-type peptide composed of a 15-amino acid diastereomeric sequence containing D- and L-forms of leucine and lysine can disrupt the plasma membrane (Non-Patent Document 19). This peptide kills tumor cells better than normal cells, and disintegrates the cell membrane in a detergent-like manner. Cell selectivity is probably determined predominantly by an increase in the level of acidic components or phosphatidylserine on the cancer cell wall (Non-Patent Document 19). The diastereomeric sequence preserves activity in serum and in the presence of proteolytic enzymes (Non-Patent Document 20). It has been suggested that the peptide's selectivity is probably influenced predominantly by an increase in the level of acidic components or phosphatidylserine on the cancer cell wall (Non-Patent Document 19). This lytic peptide has selective cytotoxicity between normal and cancer cells, but still kills normal cells at a lower concentration, and thus is not suitable for the combination with peptides with targeting moiety. Furthermore, the peptides disclosed in Documents 17, 19 and 20 are not suitable for molecular targeting, since enhancement of a cell-killing effect by EGFR targeting is not observed.
Several potential molecular-targeted anticanter marketed drugs inhibit receptor tyrosine kinase and tumor growth. In some cases, mutations of kinase-related signal molecule genes in cancer cells result in the resistance to tyrosine kinase inhibitor (TKI) drugs. Recently, it was revealed that k-ras mutations are significantly associated with a lack of response to epidermal growth factor receptor (EGFR) TKIs and cetuximab in patients with non-small-cell lung cancer and advanced colorectal cancer (Non-Patent Document 21). To overcome this critical issue, development of a novel molecular-targeted anticancer drug directly killing cancer cells, which is superior to a signal pathway blocker is desired.